Improving Whole-Cell Taxifolin Production Performance Through Growth-coupled Directed Laboratory Evolution of Flavanone 3-hydroxylase in Escherichia coli

The unique hydroxylation pattern confers dihydroflavonols with multiple beneficial functions to human health. The bioproduction of dihydroflavonols is strictly driven by flavanone 3-hydroxylase (F3H. This study focused on producing taxifolin, a typical high-value dihydroflavonol. We established taxifolin synthesis route by substituting the plant-derived flavonoid 3'-hydroxylase (F3'H) with prokaryote-derived two-component 4HPA3H system (HpaB and HpaC). Kinetics of the production indicated that F3H is the rate-limiting node. A directed library evolution (DLE) method was proposed to debottleneck this route. The tricarboxylic acid (TCA) cycle was rebuilt in Escherichia coli to couple DLE with cell growth. Very high transformation efficiency was achieved after medium optimization, thus facilitating the mutant library building. A library, theoretically containing 42360 mutants, was built and underwent a 4-day DLE. Among the randomly picked out mutants, SlF3H^v01 showed significantly accelerated catalytic rate. The whole-cell containing SlF3H^v01 produced 301.28% more taxifolin (93.82 mg/L) at 22 h and 29.73% more taxifolin (119.87 mg/L) at 46 h than that containing the wild type. This study built an E. coli chassis and developed an easy-to-manipulate DLE method to improve the taxifolin production. It can also be expanded to improve the performance of whole-cell catalyst or microbial cell factory containing other α-ketoglutarate-dependent dioxygenase.